(607g) Synthesis and Characterization of a Novel Protac Containing a ?-Hairpin Sequence Motif to Selectively Knockdown Tau Protein

Proteolysis targeting chimeras (PROTACs) have emerged as a new class of peptide-based therapeutics that utilize the ubiquitin-proteasome system (UPS) to selectively degrade a target protein. PROTACs function by harnessing the naturally occurring ubiquitylation machinery to facilitate proteasomal degradation of “undruggable” targets as an alternative approach to inhibiting an active site. Traditional protein ubiquitylation relies on an E3 ubiquitin ligase recognizing and binding to a specific sequence of amino acids on a target protein called a degron followed by transfer of the polyubiquitin cargo to a proximal lysine residue. The majority of PROTACs contain three essential components: (i) the degron, (ii) a cell penetrating peptide (to facilitate uptake into intact cells), and (iii) a binding motif for a target protein to facilitate its ubiquitylation and degradation. While PROTACs have been shown to successfully degraded numerous targets, many have been found to exhibit low cell permeability and high protease susceptibility resulting in the need for very high concentration to achieve the desired knockdown of a target protein. Previous work from our group has established a library of peptides containing a β-hairpin sequence motif that functions as both a degron and a cell penetrating peptide resulting in their rapid intracellular ubiquitylation in intact cells. Degradation studies further demonstrated that these peptides are long-lived under cytosolic conditions with a measured half-life of 420 minutes compared to unstructured CPPs, which exhibited a half-life of 10 minutes. Two members of this library RWRWR (RWRVpGRWIRQ) and OWRWR (OWRVpGRWIRQ) were rapidly internalized into cells, reaching a statistically significant permeability efficiency within 20 minutes. The goal of this study was to synthesize and test a novel class of β-hairpin-containing PROTACs that will exhibit greater stability and uptake in intact cells. The degradation of Tau protein was selected to characterize the performance of the novel PROTACs by comparing peptides with the β-hairpin motif to a previously published PROTAC using a degron from HIF-1α and a D-chirality octo-arginine CPP. The Tau protein is involved in Alzheimer’s disease and has been shown to be susceptible to PROTAC-based degradation. Using the same binding sequence for Tau, a library of novel PROTACs were synthesized incorporating either OWRWR or RWRWR and the location of the β-hairpin sequences was investigated by conjugating the peptide on the N- or C-terminus of the Tau binding sequence to determine the effect of position on protein degradation. We also investigated the necessity of adding a linker between the β-hairpin and the Tau binding region to determine if any steric issues existed. PROTAC stability was determined using a degradation assay and uptake was quantified by fluorometry. Western blotting was used to confirm knockdown of Tau in both time- and concentration-dependent studies. The results indicate that the PROTACs containing the RWRWR sequence exhibited significant degradation of Tau compared to those with the OWRWR sequence suggesting that OWRWR-based PROTACS were self-ubiquitylating. PROTACs containing the β-hairpin sequence motif had higher stability and greater uptake, suggesting their potential as a new toolkit to be incorporated into future PROTACs with the potential for enhanced performance and lifetime in cells leading to a greater degree of knockdown in target proteins.